Lactobacillus salivarius
By developing Lactobacillus salivary CGMCC No. 23518 and its fermentation broth extract, the problem of drug-resistant pathogenic bacteria proliferation was solved, effective inhibition of a variety of pathogenic bacteria was achieved, and the impact on probiotics was avoided. A safe antibacterial regimen.
Patent Information
- Application Number
- CN202411537867.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-31
- Publication Date
- 2024-12-13
AI Technical Summary
The prior art is difficult to effectively inhibit the proliferation of drug-resistant pathogenic bacteria, especially intestinal and oral pathogenic bacteria, and the use of traditional chemical preservatives and antibiotics will bring side effects and drug resistance problems.
Develop a Lactobacillus salivary CGMCC No. 23518 and its fermentation broth extract, and prepare antibacterial extracts through specific culture and treatment methods for inhibiting intestinal, oral, epidermal and environmental pathogenic bacteria, including drug-resistant microorganisms .
This antibacterial extract can effectively inhibit a variety of pathogenic bacteria and antibiotic-tolerant strains, but has no inhibitory effect on probiotics, and has thermal stability and a specific molecular weight structure.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microbial technology, and in particular to a Lactobacillus salivarius. Background Art
[0002] The rampant proliferation of environmental pathogens and spoilage bacteria is one of the main reasons for the frequent occurrence of food safety problems in recent years. These microorganisms directly or indirectly cause various harms to the human body and cause a series of diseases, such as diarrhea caused by Escherichia coli. In addition to the risk of pathogenicity from exogenous ingestion, the inherent enterococci in the human body are also an uncontrollable factor. This conditional pathogen causes bacteremia, respiratory tract and endocarditis in hosts with low immunity. In recent years, with the abuse of antibiotics, the drug resistance of enterococci has gradually increased. In some clinical infection cases, Enterococcus faecium and Enterococcus faecalis are the most common infectious bacteria. Among them, vancomycin-resistant Enterococci (VRE) are particularly common. The widespread spread of this strain around the world has had a great impact on public health and social security. In addition, a large number of pathogenic bacteria are also parasitic in the human oral cavity. Take Nocardia cavitaria as an example. It germinates and grows in a specific oral environment, eventually leading to periodontitis and odontogenic infection.
[0003] At present, the preferred option to inhibit these pathogenic microorganisms is to use chemical preservatives. However, excessive abuse of such preservative technology will lead to a large amount of residues in feed and even food, which will become one of the potential factors threatening human health. Although Nisin, a bacteriocin from Streptococcus lactis, is safe and non-toxic and avoids the residual hazards caused by chemical preservatives, the "lanthionine ring" in its molecule is also harmful to probiotics. Antibiotics are another means of inhibiting bacteria, but long-term use will cause the problem of "antibiotic resistance". Some microorganisms can even survive by withstanding strong antibiotics at the level of vancomycin, such as vancomycin-resistant Enterococcus faecium.
[0004] Lactobacillus salivarius is a gram-positive bacterium of facultative anaerobicity, which is separated from the oral cavity of human body the earliest, and is therefore one of the primary bacteria of human body. In secretions such as human intestinal tract, breast milk, vagina, etc., the existence of Lactobacillus salivarius can be detected. In recent years, various beneficial effects such as Lactobacillus salivarius stimulating immune response have been reported, but the research or report on the antibacterial effect of such microorganisms is relatively rare. Therefore, it is a problem for those skilled in the art to find a strain of Lactobacillus salivarius that has an inhibitory function to pathogenic microorganisms, especially tolerating antibiotics. Summary of the invention
[0005] In order to solve the above technical problems, the present application provides a Lactobacillus salivarius, whose preservation number is CGMCC No.23518.
[0006] The present application also provides a method for preparing a fermentation broth extract, comprising the following steps: 1) inoculating the seed liquid of the above-mentioned Lactobacillus salivarius into a liquid culture medium, culturing, and obtaining a fermentation broth; 2) centrifuging the fermentation broth to obtain a supernatant; 3) adding ammonium sulfate to the supernatant, centrifuging, and collecting a precipitate; 4) re-dissolving the precipitate with distilled water, ultrafiltering with an ultrafiltration tube, and freeze-drying the obtained macromolecules to obtain a fermentation broth extract.
[0007] The present application also provides a fermentation broth extract, which is prepared by the above-mentioned fermentation broth extract preparation method.
[0008] The present application also provides a food, medicine or health product, wherein the food, medicine or health product contains the above-mentioned Lactobacillus salivarius or the above-mentioned fermentation broth extract.
[0009] The beneficial effects brought by the present application include but are not limited to: (1) The present application discovered a strain of Lactobacillus salivarius, with the deposit number: CGMCC No. 23518. The antibacterial extract prepared using the Lactobacillus salivarius of the present application has the effect of inhibiting intestinal pathogens, oral pathogens, epidermal pathogens, environmental pathogens and antibiotic-resistant microorganisms. (2) The antibacterial extract prepared by the present application has no inhibitory effect on conventional probiotics. DETAILED DESCRIPTION
[0010] As shown in this specification and claims, unless the context clearly indicates an exception, the words "a", "an", "an" and / or "the" do not refer to the singular and may also include the plural. Generally speaking, the terms "comprise" and "include" only indicate the inclusion of the steps and elements that have been clearly identified, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements.
[0011] The present application provides a Lactobacillus salivarius, whose preservation number is CGMCC No.23518.
[0012] The deposit information of the strain in this application is as follows:
[0013] Strain name: Lactobacillus salivarius
[0014] The accession number is: CGMCC No.23518;
[0015] Deposit date: September 29, 2021;
[0016] Name of depository: General Microbiology Center of China Microbiological Culture Collection Administration;
[0017] Abbreviation of depository unit: CGMCC;
[0018] Address of the preservation unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.
[0019] In some embodiments, the 16S rRNA coding sequence of Lactobacillus salivarius may include the nucleotide sequence shown as SEQ ID NO.1.
[0020] The present application also provides a method for preparing a fermentation broth extract, comprising the following steps: 1) inoculating the seed liquid of the above-mentioned Lactobacillus salivarius into a liquid culture medium, culturing, and obtaining a fermentation broth; 2) centrifuging the fermentation broth to obtain a supernatant; 3) adding ammonium sulfate to the supernatant, centrifuging, and collecting a precipitate; 4) re-dissolving the precipitate with distilled water, ultrafiltering with an ultrafiltration tube, and freeze-drying the obtained macromolecules to obtain a fermentation broth extract.
[0021] In some embodiments, in step 1), the culture time may be 24 to 48 hours. For example, in step 1), the culture time may be 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48 hours. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0022] In some embodiments, in step 1), the culture may be anaerobic culture.
[0023] In some embodiments, in step 1), the culture temperature may be 30-40° C. For example, in step 1), the culture temperature may be 30, 32, 34, 36, 38 or 40° C. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0024] In some embodiments, in step 1), the liquid culture medium may be TPY liquid culture medium.
[0025] In some embodiments, in step 1), the effective viable count in the seed solution may be 6 to 10 x 10 8 CFU / mL. For example, in step 1), the effective viable count in the seed solution can be 6, 7, 8, 9 or 10x10 8 CFU / mL. It also includes any range characterized by a combination of the above end values, which will not be repeated here. In some embodiments, preferably, in step 1), the effective viable count in the seed solution can be 8x10 8 CFU / mL.
[0026] In some embodiments, in step 1), the volume ratio of the seed solution to the liquid culture medium may be 1-5%. For example, the volume ratio of the seed solution to the liquid culture medium may be 1, 2, 3, 4 or 5%. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0027] In some embodiments, in step 2), the centrifugation time may be 10 to 20 minutes. For example, in step 2), the centrifugation time may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 minutes. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0028] In some embodiments, in step 2), the centrifugal speed may be 8000-12000 g. For example, in step 2), the centrifugal speed may be 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500 or 12000 g. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0029] In some embodiments, in step 3), the saturation of ammonium sulfate in the supernatant can be 30-70%. For example, in step 3), the saturation of ammonium sulfate in the supernatant can be 30, 40, 50, 60 or 70%. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0030] In some embodiments, in step 3), the centrifugation time may be 10 to 20 min. For example, in step 3), the centrifugation time may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 min. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0031] In some embodiments, in step 3), the centrifugal speed may be 8000-12000 g. For example, in step 3), the centrifugal speed may be 8000, 8500, 9000, 9500, 10000, 10500, 11000, 11500 or 12000 g. Any range characterized by a combination of the above end values is also included, which will not be repeated here.
[0032] In some embodiments, in step 4), the ultrafiltration tube molecular weight cutoff can be 3 kDa.
[0033] The present application also provides a fermentation broth extract, which is prepared by the above-mentioned fermentation broth extract preparation method.
[0034] In some embodiments, the fermentation broth extract is thermostable.
[0035] In some embodiments, the molecular weight of the antibacterial substance in the fermentation broth extract may be greater than 3000 Da.
[0036] The present application also provides a food, medicine or health product, wherein the food, medicine or health product contains the above-mentioned Lactobacillus salivarius or the above-mentioned fermentation broth extract.
[0037] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.
[0038] The strain information used in this application is as follows:
[0039] Vancomycin-resistant Enterococcus faecium CICC 24261, purchased from CICC, China;
[0040] Staphylococcus epidermidis CICC 10294, purchased from CICC, China;
[0041] Pseudomonas fluorescens CICC 23250, purchased from CICC, China;
[0042] Lactiplantibacillus plantarum CGMCC 0847, provided by Bright Dairy Co., Ltd.;
[0043] Bifidobacterium animalis subsp. Lactis CGMCC 1.15623, purchased from CGMCC, China;
[0044] Lactobacillus rhamnosus CGMCC 13310, provided by Bright Dairy Co., Ltd.;
[0045] Limosilactobacillus fermentum CGMCC 17321, provided by Bright Dairy Co., Ltd.;
[0046] Lactobacillus paracasei ATCC 334, purchased from ATCC, USA;
[0047] Lactiplantibacillus plantarum ATCC 14917, purchased from ATCC, USA;
[0048] Streptococcus thermophilus CGMCC 1.8748, purchased from CGMCC, China;
[0049] Salmonella enterica CGMCC 1.10754, purchased from CGMCC, China;
[0050] Listeria Monocytogenes CGMCC 1.10753, purchased from CGMCC, China;
[0051] Nocardia coeliaca ATCC 17041, purchased from ATCC, USA;
[0052] Staphylococcus aureus CGMCC 1.2465, purchased from CGMCC, China;
[0053] Escherichia coli ATCC 25922 was purchased from ATCC, USA.
[0054] Example 1 - Screening, Identification and Preservation of Lactobacillus salivarius PC114
[0055] The Lactobacillus salivarius PC114 in the present invention is obtained by the following method: aseptically weighing 1 g of stool sample from a long-lived elderly population, serially diluting with sterile physiological saline, uniformly coating the dilution on a sterile MRS agar medium (purchased from Merck, Germany) by coating, anaerobically culturing at 37° C. for 48 hours, selecting a single white colony with a smooth colony edge and streaking it again on the MRS agar medium, and after anaerobically culturing at 37° C. for 24 hours, selecting a single colony and entrusting the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration to perform 16S rRNA gene sequencing and physiological and biochemical tests (as shown in Table 1).
[0056] The 16S rRNA gene sequence of this strain is as follows:
[0057]
[0058] Table 1 Results of physical and chemical tests on strains
[0059] Test Project result Test Project result Test Project result glycerin - Inositol - Inulin - Erythrocarb - Mannitol + Melezitose - D-arabinose - Sorbitol + Raffinose + L-arabinose - α-Methyl-D-mannoside - starch - D-Ribose - α-Methyl-D-glucoside - Glycogen - D-Xylose - N-acetyl-glucosamine + Xylitol - L-Xylose - Amygdalin - Gentianobiose - Adonol - Arbutin - D-turanose - β-Methyl-D-xyloside - Aesculin - D-Lyxose - D-Galactose + Salicin - D-Tagatose - D-Glucose + Cellobiose - D-fucose - D-Fructose + maltose + L-fucose + D-Mannose - lactose + D-arabinitol + L-sorbitol - Melibiose + L-arabinitol - L-Rhamnose - sucrose + Gluconate - Dacron - Trehalose + 2-Keto-gluconate -
[0060] The strain was identified as Lactobacillus salivarius by 16S rRNA gene sequence comparison and physiological and biochemical analysis at the China General Microbiology Center (CGMCC). The strain was deposited at the China General Microbiology Center (CGMCC) on September 29, 2021. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101. The deposit number of the strain is: CGMCC No. 23518.
[0061] Example 2 - Application of Lactobacillus salivarius PC114 in the preparation of antibacterial extracts
[0062] 1. Materials and methods
[0063] Preparation of fermentation seed liquid of Lactobacillus salivarius PC114
[0064] The lyophilized powder of Lactobacillus salivarius PC114 was dissolved with a small amount of sterile distilled water, and a loop was streaked on MRS solid medium (purchased from Merck, Germany) with an inoculation loop, and anaerobically cultured at 37°C for 48 h. A single colony was picked with an inoculation loop and placed in 10 mL MRS liquid medium (purchased from Merck, Germany). The colony was evenly dispersed in the liquid medium using a vortex oscillator, cultured at 37°C for 24 h, and inoculated into MRS liquid medium with an inoculum of 2% (v / v). After culturing again at 37°C for 24 h, the culture was centrifuged at 15,000 rpm for 10 minutes, the supernatant was discarded, the bacteria were washed twice with sterile distilled water, and then suspended with sterile distilled water of the original culture volume to obtain the fermentation seed liquid of Lactobacillus salivarius PC114. The viable count of the seed liquid was determined to be 8×10 8 CFU / mL.
[0065] 2. Preparation of antibacterial extract of Lactobacillus salivarius PC114
[0066] The seed liquid of Lactobacillus salivarius PC114 was aseptically inoculated into TPY liquid medium at an inoculum amount of 3% (v / v), and the fermentation liquid was obtained by anaerobically culturing at 36°C for 36 hours, and the fermentation liquid was centrifuged at 10,000g for 15 minutes to obtain the supernatant. Ammonium sulfate was slowly added to the supernatant, and the precipitate was collected by centrifugation at 10,000g for 15 minutes when the ammonium sulfate saturation was 30% to 70%, and the precipitate was re-dissolved with distilled water and then ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part was freeze-dried to obtain the antibacterial extract A of Lactobacillus salivarius PC114.
[0067] Example 3 - Application of Lactobacillus salivarius PC114 in the preparation of antibacterial extracts
[0068] 1. Materials and methods
[0069] Preparation of fermentation seed liquid of Lactobacillus salivarius PC114: Same as Example 2.
[0070] 2. Preparation of antibacterial extract of Lactobacillus salivarius PC114
[0071] The seed liquid of Lactobacillus salivarius PC114 is aseptically inoculated in a TPY liquid medium at an inoculum amount of 1% (v / v), and the fermentation liquid is obtained by anaerobically culturing at 40°C for 48 hours, and the fermentation liquid is centrifuged at 12,000g for 10 minutes to obtain the supernatant. Ammonium sulfate is slowly added to the supernatant, and the precipitate is collected by centrifugation at 12,000g for 10 minutes when the ammonium sulfate saturation is 60% to 70%, and the precipitate is re-dissolved with distilled water and then ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part is freeze-dried to obtain the antibacterial extract B of Lactobacillus salivarius PC114.
[0072] Example 4 - Application of Lactobacillus salivarius PC114 in the preparation of antibacterial extracts
[0073] 1. Materials and methods
[0074] Preparation of fermentation seed liquid of Lactobacillus salivarius PC114: Same as Example 2.
[0075] 2. Preparation of antibacterial extract of Lactobacillus salivarius PC114
[0076] The seed liquid of Lactobacillus salivarius PC114 was aseptically inoculated in a TPY liquid medium at an inoculum amount of 5% (v / v), and the fermentation liquid was obtained by anaerobically culturing at 30°C for 30 hours, and the fermentation liquid was centrifuged at 8,000g for 20 minutes to obtain the supernatant. Ammonium sulfate was slowly added to the supernatant, and the precipitate was collected at an ammonium sulfate saturation of 40% to an ammonium sulfate saturation of 60% by centrifugation at 8,000g for 20 minutes, and the precipitate was reconstituted with distilled water and then ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part was freeze-dried to obtain the antibacterial extract C of Lactobacillus salivarius PC114.
[0077] Example 5 - Application of Lactobacillus salivarius PC114 in the preparation of antibacterial extracts
[0078] 1. Materials and methods
[0079] Preparation of fermentation seed liquid of Lactobacillus salivarius PC114: Same as Example 2.
[0080] 2. Preparation of antibacterial extract of Lactobacillus salivarius PC114
[0081] The seed liquid of Lactobacillus salivarius PC114 was aseptically inoculated into TPY liquid medium at an inoculum amount of 2% (v / v), and the fermentation liquid was obtained by anaerobically culturing at 38°C for 24 hours, and the fermentation liquid was centrifuged at 11,000g for 13 minutes to obtain the supernatant. Ammonium sulfate was slowly added to the supernatant, and the precipitate was collected at the ammonium sulfate saturation of 50% to 70% by centrifugation at 11,000g for 13 minutes, and the precipitate was reconstituted with distilled water and then ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part was freeze-dried to obtain the antibacterial extract D of Lactobacillus salivarius PC114.
[0082] Example 6 - Application of Lactobacillus salivarius PC114 in the preparation of antibacterial extracts
[0083] 1. Materials and methods
[0084] Preparation of fermentation seed liquid of Lactobacillus salivarius PC114: Same as Example 2.
[0085] 2. Preparation of antibacterial extract of Lactobacillus salivarius PC114
[0086] The seed liquid of Lactobacillus salivarius PC114 was aseptically inoculated in a TPY liquid medium at an inoculum amount of 4% (v / v), and the fermentation liquid was obtained by anaerobically culturing at 33°C for 42 hours, and the fermentation liquid was centrifuged at 9,000g for 17 minutes to obtain the supernatant. Ammonium sulfate was slowly added to the supernatant, and the precipitate was collected at an ammonium sulfate saturation of 50% to an ammonium sulfate saturation of 60% by centrifugation at 9,000g for 17 minutes, and the precipitate was reconstituted with distilled water and then ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part was freeze-dried to obtain the antibacterial extract E of Lactobacillus salivarius PC114.
[0087] Effect Example 1 - Inhibitory Effect of Antibacterial Extract of Lactobacillus salivarius PC114 on Probiotics
[0088] 1. Materials and methods
[0089] (a) Preparation of probiotic suspension: Lactobacillus plantarum CGMCC 0847, Lactobacillus paracasei ATCC334, Bifidobacterium CGMCC 1.15623, Lactobacillus rhamnosus CGMCC 13310 and Lactobacillus fermentum CGMCC The lyophilized powder of 17321 was dissolved with a small amount of sterile distilled water, and a loop was taken with an inoculating loop and streaked on TPY solid culture medium (purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., China), and cultured at 37°C for 48 hours, and a single colony was picked with an inoculating loop and placed in 10 mL TPY liquid culture medium (purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd., China). The colony was evenly dispersed in the liquid culture medium using a vortex oscillator, cultured at 37°C for 24 hours, and inoculated into TPY liquid culture medium with an inoculum of 2% (v / v). After culturing again at 37°C for 24 hours, the culture was centrifuged at 15,000 rpm for 10 minutes, the supernatant was discarded, the bacteria were washed twice with sterile distilled water, and then suspended with sterile distilled water of the original culture volume to obtain a bacterial suspension of each indicator bacteria.
[0090] (b) Preparation of indicator bacteria plates
[0091] The indicator bacteria suspension prepared by the above method was diluted to 10 6 CFU / mL, take appropriate amount of bacterial solution and evenly spread it on TPY plate, then you can get indicator bacteria plate containing different probiotics
[0092] 2. Inhibitory effect of Lactobacillus salivarius PC114 antibacterial extract on probiotics
[0093] An Oxford cup was placed on the above-mentioned probiotic indicator bacteria plate, and 100 μL of the antibacterial extract solution A prepared in the above-mentioned Effective Example 1 was added to each Oxford cup. The cups were incubated at 37° C. for 2 days, and the diameter of the antibacterial zone was measured. The results are shown in Table 2 below.
[0094] Table 2 Inhibition results of Lactobacillus salivarius PC114 antibacterial extract on probiotics
[0095] Diameter of inhibition zone (mm) Lactobacillus salivarius PC114 antibacterial extract solution A Lactobacillus plantarum - Lactobacillus paracasei - Bifidobacterium - Lactobacillus rhamnosus - Lactobacillus mucilaginosus fermentation -
[0096] As shown in Table 2 above, the antibacterial extract solution of Lactobacillus salivarius PC114 has no inhibitory effect on probiotics including Lactobacillus plantarum, Lactobacillus paracasei, Bifidobacterium, Lactobacillus rhamnosus and Lactobacillus fermentum.
[0097] Effect Example 2 - Antibacterial Effect of Antibacterial Extract of Lactobacillus salivarius PC114
[0098] (a) Preparation of indicator bacteria suspension: Referring to the preparation method of the probiotic suspension described in Example 1, bacterial suspensions of Salmonella CGMCC 1.10754, Listeria monocytogenes CGMCC 1.10753, Nocardia vacuole ATCC 17041, Staphylococcus aureus CGMCC 1.2465, Escherichia coli ATCC 25922, vancomycin-resistant Enterococcus faecium CICC 24261, Staphylococcus epidermidis CICC 10294 and Pseudomonas fluorescens CICC 23250 were prepared respectively.
[0099] (b) Preparation of indicator bacteria plates: Referring to the method described in Example 1, the indicator bacteria suspension was used to prepare indicator bacteria plates containing different pathogenic bacteria.
[0100] (c) Antibacterial effect of Lactobacillus salivarius PC114 antibacterial extract
[0101] The antibacterial extracts A, B, C, D and E of Lactobacillus salivarius PC114 prepared above were dissolved in sterile water to obtain the corresponding antibacterial extract solutions (15 mg / mL). The antibacterial extract solutions were placed on the indicator bacteria plates previously inoculated with pathogenic bacteria using the Oxford cup method (100 uL / cup), and cultured at 37° C. for 2 days. The diameter of the inhibition zone was measured, and the results are shown in Table 3 below.
[0102] Table 3 Inhibition results of Lactobacillus salivarius PC114 antibacterial extract on pathogenic bacteria
[0103]
[0104] It can be seen from Table 3 above that the antibacterial zone diameter of the antibacterial extract of Lactobacillus salivarius PC114 against intestinal pathogens is ≥14 mm, the antibacterial zone diameter against oral pathogens is ≥20 mm, the antibacterial zone diameter against epidermal pathogens is ≥8 mm, the antibacterial zone diameter against environmental pathogens is ≥9 mm, and the antibacterial zone diameter against antibiotic-resistant microorganisms is ≥19 mm.
[0105] Comparative Example 1 - Inhibitory effects of fermentation broth extracts of different strains on pathogenic bacteria
[0106] Referring to the method described in Example 2, the inhibitory effects of TPY fermentation broth extracts prepared from Lactobacillus salivarius PC114, Lactobacillus paracasei ATCC 334, Lactobacillus plantarum ATCC 14917, and Streptococcus thermophilus CGMCC 1.8748 on different pathogens were compared. The specific operations were as follows:
[0107] 1. Materials and methods
[0108] (a) Preparation of seed solution or bacterial suspension: Seed solution or bacterial suspension of Lactobacillus salivarius PC114, Lactobacillus paracasei ATCC 334, Lactobacillus plantarum ATCC 14917, Streptococcus thermophilus CGMCC 1.8748 and pathogenic bacteria (Salmonella CGMCC 1.10754, Listeria monocytogenes CGMCC 1.10753, Nocardia vacuole ATCC17041, Staphylococcus aureus CGMCC 1.2465, Escherichia coli ATCC 25922, vancomycin-resistant Enterococcus faecium CICC24261, Staphylococcus epidermidis CICC 10294 and Pseudomonas fluorescens CICC 23250) were prepared respectively according to the method described in Example 1.
[0109] (b) Preparation of indicator bacteria plates
[0110] Referring to the method described in Effect Example 1, the above pathogenic bacteria suspension was used to prepare indicator bacteria plates containing different pathogenic bacteria.
[0111] (c) Preparation of fermentation broth extracts of different strains
[0112] The seed liquid of Lactobacillus salivarius PC114, Lactobacillus paracasei ATCC 334, Lactobacillus plantarum ATCC 14917 and Streptococcus thermophilus CGMCC 1.8748 is aseptically inoculated in a TPY liquid medium at an inoculum amount of 3% (v / v), anaerobically cultured at 36°C for 36 hours to obtain a fermentation liquid, centrifuged at 10,000g for 15 minutes, and the supernatant is taken. Ammonium sulfate is slowly added to the supernatant, and the precipitate precipitated when the ammonium sulfate saturation is 30-70% is collected by centrifugation at 10,000g for 15 minutes, and then re-dissolved with a small amount of distilled water and ultrafiltered and desalted with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and the macromolecular part is freeze-dried to obtain the fermentation extract of the corresponding strain.
[0113] 2. Inhibitory effects of fermentation extracts of different strains on pathogenic bacteria
[0114] An Oxford cup was placed on the above pathogenic bacteria indicator bacteria plate, and 100 μL of the fermentation extract solution (15 mg / mL) of the different strains prepared above was added to each Oxford cup. The culture was incubated at 37° C. for 2 days, and the diameter of the inhibition zone was measured. The results are shown in Table 4 below.
[0115] Table 4 Inhibition results of fermentation extracts of different strains on pathogenic bacteria
[0116]
[0117] It can be seen from Table 4 above that other TPY fermentation strains do not have the ability to ferment TPY to produce antibacterial substances, while Lactobacillus salivarius PC114 can utilize TPY fermentation to synthesize specific products that inhibit various pathogenic bacteria.
[0118] The basic concepts have been described above. Obviously, for those skilled in the art, the above detailed disclosure is only for example and does not constitute a limitation of this specification. Although not explicitly stated here, those skilled in the art may make various modifications, improvements and corrections to this specification. Such modifications, improvements and corrections are suggested in this specification, so such modifications, improvements and corrections still belong to the spirit and scope of the exemplary embodiments of this specification.
[0119] At the same time, this specification uses specific words to describe the embodiments of this specification. For example, "one embodiment", "an embodiment", and / or "some embodiments" refer to a certain feature, structure or characteristic related to at least one embodiment of this specification. Therefore, it should be emphasized and noted that "one embodiment" or "an embodiment" or "an alternative embodiment" mentioned twice or more in different positions in this specification does not necessarily refer to the same embodiment. In addition, certain features, structures or characteristics in one or more embodiments of this specification can be appropriately combined.
[0120] In some embodiments, numbers describing the number of components and attributes are used. It should be understood that such numbers used in the description of the embodiments are modified by the modifiers "about", "approximately" or "substantially" in some examples. Unless otherwise specified, "about", "approximately" or "substantially" indicate that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may change according to the required features of individual embodiments. In some embodiments, the numerical parameters should take into account the specified significant digits and adopt the general method of retaining digits. Although the numerical domains and parameters used to confirm the breadth of their range in some embodiments of this specification are approximate values, in specific embodiments, the setting of such numerical values is as accurate as possible within the feasible range.
[0121] Finally, it should be understood that the embodiments described in this specification are only used to illustrate the principles of the embodiments of this specification. Other variations may also fall within the scope of this specification. Therefore, as an example and not a limitation, alternative configurations of the embodiments of this specification may be considered consistent with the teachings of this specification. Accordingly, the embodiments of this specification are not limited to the embodiments explicitly introduced and described in this specification.
Claims
1. A Lactobacillus salivarius, whose preserving number is CGMCC No.23518.
2. Lactobacillus salivarius as claimed in claim 1, It is characterized in that The 16S rRNA coding sequence of Lactobacillus salivarius includes the nucleotide sequence shown in SEQ ID NO.
1.
3. A method for preparing a fermentation broth extract, The following steps are involved: 1) inoculating the seed liquid of Lactobacillus salivarius according to claim 1 or 2 into a liquid culture medium, culturing, and obtaining a fermentation liquid; 2) centrifuging the fermentation broth to obtain a supernatant; 3) adding ammonium sulfate to the supernatant, centrifuging, and collecting the precipitate; 4) re-dissolving the precipitate with distilled water, ultrafiltration with an ultrafiltration tube, and freeze-drying the obtained macromolecules to obtain a fermentation broth extract.
4. The preparation method according to claim 3, It is characterized in that The step 1) includes the following features: The culture time is 24 to 48 hours; And / or, the culture is anaerobic culture; And / or, the culture temperature is 30-40°C; And / or, the liquid culture medium is TPY liquid culture medium; And / or, the effective viable bacteria count in the seed solution is 6 to 10 x 10 8 CFU / mL; The volume ratio of the seed liquid to the liquid culture medium is 1-5%.
5. The preparation method according to claim 3, It is characterized in that The step 2) includes the following features: The centrifugation time is 10 to 20 minutes; And / or, the centrifugal speed is 8000-12000g.
6. The preparation method according to claim 3, It is characterized in that The step 3) includes the following features: The saturation of ammonium sulfate in the supernatant is 30-70%; And / or, the centrifugation time is 10 to 20 minutes; And / or, the centrifugal speed is 8000-12000g.
7. The preparation method according to claim 3, It is characterized in that In the step 4), the molecular weight cut-off of the ultrafiltration tube is 3 kDa.
8. A fermentation broth extract obtained by the method for preparing the fermentation broth extract according to any one of claims 3 to 7.
9. The fermentation broth extract according to claim 8, It is characterized in that The fermentation broth extract is thermally stable; And / or, the molecular weight of the antibacterial substance in the fermentation broth extract is greater than 3000Da.
10. A food, medicine or health product, It is characterized in that The food, medicine or health product contains the Lactobacillus salivarius according to claim 1 or 2 or the fermentation broth extract according to claim 8 or 9.